Favipiravir (FVR) is a repurposed antiviral drug for treating mild to moderate cases of the novel coronavirus disease 2019 (COVID-19). However, its poor solubility and permeability limit its clinical efficacy. To overcome its physicochemical and pharmacokinetic limitations, we statistically designed a mucoadhesive chitosan–alginate nanoparticles (MCS-ALG-NPs) as a new carrier for FVR using response surface methodology, which provided suitable characteristics for transmucosal delivery. The use of mucoadhesive polymers for intranasal administration promotes the residence time and contact of FVR in the mucus membrane. The optimized FVR-MCS-ALG-NPs demonstrated superior mucoadhesion, higher permeation and deposition in the nasal mucosa, and a significant increase in the inhibition of viral replication over 35-fold compared with free FVR. The overall results suggest that MCS-ALG-NPs could be used as an effective mucoadhesive carrier to enhance the activity of FVR against COVID-19. The novel coronavirus disease 2019 (COVID-19) remains a primary global health concern due to its high infectivity and mortality rate. Its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) commonly spreads through the inhalation of airborne droplets or direct contact with the virus in the mucosa . FortunaTo further improve medication efficacy and targeted drug delivery, the administration of therapeutics through the nose, which is also the primary transmission route of SARS-CoV-2, appears to be suitable for achieving high localization of antivirals. Intranasal delivery has already been used in various respiratory viral infections as it provides either local or systemic effects proximal to infection. It also offers several advantages over systemic delivery systems because of its non-invasiveness, fast onset of action, and in various cases can reduce the side effects of the drug through its direct and targeted delivery approach . In addiβ-(1-4) glycosidic bonds, respectively, made it more favorable for nasal physiological conditions (pH 5.5 and the presence of lysozyme at ~30 µg/mL) [Thus, the development of NPs using mucoadhesive biopolymers can effectively overcome these mucosal barriers and provide high drug deposition in the nasal cavities. Mucoadhesion emerged as a strategy that promotes sustained release, improved residence time via their interaction with the mucus for immobilization, and enhanced site-specificity and cellular uptake . Positiv0 µg/mL) . Pyrc et0 µg/mL) . However0 µg/mL) . ALG has0 µg/mL) .In theory, direct local therapies to the upper respiratory tract where the highest viral load could be found may provide an alternative way to potentially reduce the viral loads and transmission of SARS-CoV-2. However, data on the encapsulation of antiviral agents, especially FVR, and their nasal delivery for respiratory infections are limited. In a previous study, the possible mechanism associated with anti-SARS-CoV-2 infection of antiviral agent-loaded nanoformulation was postulated as the prevention of viral adsorption and invasion into the host cell through its interference with the viral envelope, leading to cellular uptake enhancement and eventually viral replication inhibition by targeting the RdRp. As such, we anticipate that the MCS-ALG-NPs can improve the physicochemical properties of FVR and effectively inhibit SARS-CoV-2 replication. The mucoadhesive property of the carrier also permits the transmucosal permeation and deposition of FVR in the nasal mucosa to directly target localized viruses in the upper airway. To achieve the desired characteristics of the FVR-MCS-ALG-NPs for the transmucosal delivery, this study used Box–Behnken design (BBD) and response surface methodology (RSM). Mucoadhesive, in vitro release and ex vivo transmucosal studies of optimized FVR-MCS-ALG-NPs were also performed. Finally, its antiviral activity against porcine epidemic diarrhea virus (PEDV) was evaluated. Herein, we demonstrated a well-tolerated and efficient mucoadhesive and muco-penetrating nanocarrier for FVR with a high permeation rate and deposition in the nasal mucosa while subsequently enhancing its anti-coronavirus effect by 35-fold.Favipiravir  was purchased from Aurore Pharmaceuticals . Chitosan  of 73 kDa, degree of deacetylation of 91.7%, and polydispersity index (PDI) of 3.8) was supplied by Marine Bio-Resources . Sodium alginate , Poloxamer-407, and porcine mucin (type III) were purchased from Sigma-Aldrich, Inc. . All chemicals used in this study were analytical grade without further purification.w/v). The solution was magnetically stirred at 1500 rpm for 20 min followed by sonication to allow FVR to completely emulsify. Then, an aqueous CaCl2 solution  was added dropwise at a rate of 20 mL/h using an automatic syringe pump. After 30 min of stirring, the CS solution  was added to the pre-gelled calcium–ALG solution and continuously stirred for another 30 min. The suspension was equilibrated overnight in a light-proof cabinet at ambient room temperature. To optimize the fabrication process and determine the effect of various factors on the size (Y1), ζ-potential (Y2), loading capacity (LC) (Y3), and encapsulation efficiency (EE) (Y4) of the FVR-MCS-ALG-NPs, a three-factor BBD was used based on the preliminary experimental data gathered considering the ALG:CS (A), FVR (B), and poloxamer 407 (C) as the independent variables  with three replicated center points to limit the error , and the ζ-potential was determined by electrophoretic mobility of the NPs, using a Zetasizer  without further dilution or pH adjustments (pH 5.5 ± 0.3). The morphology was observed by TEM  and FE-SEM . LC and EE of the FVR-MCS-ALG-NPs were determined using an indirect method after ultracentrifugation at 4 °C and 105,000 × g speed for 1 h. The absorbance was measured at 363 nm, and the amount of the free FVR from the supernatant was calculated against the standard curve . The LC (Equation (1)) and EE (Equation (2)) of the FVR-MCS-ALG-NPs were calculated as follows:®, Netzsch, Germany) in a nitrogen atmosphere at 20 °C/min. The crystalline structure was analyzed using a wide-angle X-ray diffractometer , operated at a speed of 0.2° 2θ/step at room temperature.The thermal properties were determined using a thermogravimetric analyzer  to mimic the mucus layer and nasal epithelium environment, respectively. The medium was maintained at 37 °C and stirred at 100 rpm. At pre-determined time points, an aliquot was withdrawn and centrifuged at 4000 rpm. The amount of free FVR from the supernatant was calculated against the standard curve of FVR. The cumulative FVR released was computed using Equation (4).CR (%) is cumulative FVR released, eV is collection volume (mL), oV is the volume of the medium (mL), nC is the concentration of the time point (mg/mL), and m is total FVR (mg) in the dialysis bag.Ready-to-use SnakeSkin™ dialysis tubing  was used as a membrane for the study. The sealed tubes were then submerged in 500 mL of PBS (pH 7.4) and simulated nasal fluid (SNF)  was plotted as a function of time (min). After the experiment, the FVR retained in the donor compartment was quantified. The flux at steady state (Jss) and permeability coefficient (KP) were estimated using Equations (5) and (6), respectively.2) is the diffusion area, and C0 (µg) is the initial drug concentration in the donor compartment. To determine the effect on the permeability compared to the free FVR solution, the enhancement ratio of the flux (ERflux) was computed using Equation (7):Porcine nasal mucosa (PNM) was collected from the noses of freshly slaughtered pigs in the local slaughterhouse and was processed as described in v/v) fetal bovine serum (FBS) at 37 °C in a 95% air/5% CO2 incubator. The diluents were used as the control to observe the cytotoxic effect of the test samples.The biocompatibility studies were performed in human nasal cells and porcine tissue. RPMI 2650 human nasal epithelial cells  were cultivated in MEM media supplemented with 10%  were used as the control to observe the antiviral effect of the test samples. At 18 h post-infection, the mCherry (561ex and 570–630em nm) and eGFP (488ex and 500–550em nm) fluorescent images were obtained using the Opera Phenix High-Content Screening System . The total mCherry fluorescent intensity was quantified by Harmony high-content imaging and analytical software . The relative decrease in mCherry intensity of the test samples was calculated by comparison with the control.The mCherry-PEDV was added to the seeded eGFP-Vero cells to allow viral absorption for 1 h. The unattached virus was removed by washing with PBS. The test samples were added to the cells with fresh media supplemented with 1% tryPLE. Sterile water or blank NP-treated cells (0.5% 50) was determined through a non-linear regression-curve-fit analysis. The difference between groups was compared by ANOVA followed by a postoperative multiple comparison test in GraphPad® Prism software 8.3.0.  A p value of <0.05 was considered significant.The experiments are expressed as mean ± standard deviation (SD). The half-maximal effective concentration  owing to the low electrostatic affinity of the polymers leading to instability of the system. Furthermore, the effects of the drug concentration, especially hydrophobic molecules, along with the surfactant concentration play an important role in the formulation . This is® software  to find a suitable model for optimization. ANOVA was utilized in this study to compare the variation resulting from the change in the combination of variable levels and the variation brought by the random errors in the measurements of the generated responses. The result of the statistical analysis of the mathematical model is summarized in 2 and predicted R2 must be high and in reasonable agreement (<0.2 difference), and (4) the selected model must have high adequate precision (>4 is acceptable).All 15 formulations were prepared successfully as a clear solution without any visible signs of separation or precipitation prior to characterization. The summary of the experimental runs and their corresponding responses are shown in The fit summary of the responses and the selected model are presented in The optimal conditions were computed based on searches for a combination of factor levels that would satisfy the desired goals for each response and independent factor. The goal for each factor and response are shown in Smaller particles have a higher surface area that is advantageous for absorption and transport across the nasal cavity. The SEM a–d and T2CO3, and the total decomposition of the polymer [Additionally, thermal analyses of FVR showed only a single drop at 238.0 °C attributed to the decomposition of FVR. Furthermore, three-step weight losses were observed in the TGA/DTG curve of the blank-MCS-ALG-NPs  due to t polymer . The sam polymer . Broad pp = 0.0170) after 1 h incubation with mucin (p = 0.0036) owing to the adsorption of mucin on the NP surface  changed significantly in ζ-potential (th mucin A suggest surface B. Moreovp < 0.0001) (p < 0.0001). The statistical analysis revealed no significant change in the % of mucin adsorbed after 10 min to 1 h incubation of FVR-ALG-NP samples indicating saturation of mucin binding on the NPs. Moreover, the % mucin binding in the FVR-MCS-ALG-NPs showed a gradual increase up to 30 min of incubation.The % mucin binding onto the NPs was also investigated to better understand its mucoadhesive properties. Significant mucin binding was observed in the first 10 min of incubation, wherein FVR-MCS-ALG-NPs (23.0 ± 7.0%) had more significant mucin adsorption compared to the FVR-ALG-NPs (10.0 ± 4.4%) ( 0.0001) C. After Formulations with and without CS were compared to observe the benefit of a CS coating on the release of FVR from the NPs A,B. The 2 value must be close to 1, (2) the model selection criteria (MSC) must be high, and (3) the Akaike information criterion (AIC) must be low [n” value: (1) n ≤ 0.5, the drug diffusion from the polymer matrix corresponds to a Fickian diffusion or a quasi-Fickian diffusion mechanism. (2) 0.5 < n < 1, an anomalous, non-Fickian drug diffusion occurs. (3) n = 1, a non-Fickian, case of II  transport or zero-order release kinetics could be observed, and (4) n > 1, super case II transport was observed [n” value from 0.19 to 0.23 depicts a Fickian diffusion mechanism of FVR release from the degradation of the polymer matrix  had superior permeation through the membrane with an area of 1.77 cm2 compared to the uncoated FVR-ALG-NPs (66.5 ± 11.8 µg/cm2) and free FVR (22.3 ± 13.8 µg/cm2) (p < 0.0001). The FVR-MCS-ALG-NPs had a significant permeation activity through PNM compared to the FVR-ALG-NPs (p < 0.0001). In addition, a significant amount of the FVR from the FVR-MCS-ALG-NPs and FVR-ALG-NPs was deposited in tissues compared to free FVR. Interestingly, the FVR retained (p = 0.0834) was similar to both coated and uncoated FVR-NPs. This can be explained by the slower permeation of the uncoated FVR-ALG-NPs that causes the deposition of FVR in the mucosa  mucosal specimens from each treatment group. FVR-MCS-ALG-NPs (133.2 ± 11.0 µg/cm µg/cm2) A,B. Due e mucosa A,C. The v/v of the diluent was normalized as the final concentration for the dilution in subsequent experiments  for FVR-NPs as compared to the control cells B. It is 2O) and blank-MCS-ALG-NPs was first tested to determine the possible toxicity of the diluent in eGFP-Vero cells as described in As SARS-CoV-2 is classified as a risk group 3 pathogen that requires extremely stringent safety precautions and specialized laboratory facilities, we employed non-infectious coronavirus as a surrogate for the antiviral studies. The porcine epidemic diarrhea virus  was selected as a model due to its low health risk and minimal laboratory requirements. It has biophysical properties and genomic structures similar to human coronaviruses, such as SARS-CoV-2 . Previou50 of 6.63 ± 2.42 μg/mL calculated by GraphPad® Prism software 8.3.0 (p < 0.0001), while free FVR did not have any significant reduction in PEDV replication up to 46.0 μg/mL (p > 0.9). To further determine the antiviral activity of free FVR, we investigated the samples in higher concentrations that were non-toxic to the eGFP-Vero cells (50 6.63 ± 2.42 vs. >230 μg/mL).The anti-viral effect of FVR samples was performed in three independent experiments . The stire 8.3.0 c. The poro cells . We founSeveral strategies have been employed to overcome the poor solubility and permeability of FVR from chemical synthesis to formulation design. Nanotechnology using solid-lipid NPs (SLN) has proven to enhance the antiviral effect of FVR towards SARS-CoV-2. Still, issues about the physical characteristics of the fabricated SLN and the instability of the lipid overtime call for the need to explore another suitable carrier for the drug. In this study, we introduced the concept of using polymers as a biomaterial in constructing a nanocarrier for hydrophobic drugs owing to their tunable properties concerning size, surface charge, LC, and EE that are imperative in the delivery, efficacy, and stability of the product. We also demonstrated the concept of using the intranasal route of delivering FVR as it mimics the natural transmission of the SARS-CoV-2 virus. The use of natural biocompatible carbohydrate polymers, such as CS and ALG, as the materials for the construction of nanocarriers could be a rational approach for producing an efficient and stable carrier. Moreover, their tunable size, surface characteristics, and mucoadhesive properties make them suitable for transmucosal delivery. The proposed delivery route for FVR could potentially reduce the viral load in the infected individuals and could eventually mitigate the risk of viral transmission.Here, we first demonstrate the optimization of the FVR-MCS-ALG-NPs by statistical analysis using RSM based on BBD of experiments in developing an ideal NP formulation for transmucosal delivery. The model employed had high predictability for the critical parameters with the highest desirability of 93% with respect to the goal set for the size, surface charge, LC, and EE. In investigating the effects and interaction between the major factors and responses, we found that the CS mass ratio to ALG majorly contributes to the optimization of FVR-MCS-ALG-NPs. Increasing the concentration of CS in the system contributes to the formation of larger particles brought by the presence of excess CS molecules causing the clumping and entanglement of the molecules . CS also2+, followed by the swelling and erosion of the polymer matrix and, eventually, the release of FVR [Additionally, we investigated the benefit of CS-coating onto the NPs regarding drug release, mucoadhesive, and transmucosal permeation by comparing it with the uncoated-NPs (FVR-ALG-NPs) and free FVR. Here, we demonstrated sustained release of the FVR-MCS-ALG-NPs in both pH conditions . Notably, the MCS-ALG-NPs had a higher release rate in the epithelium environment than in the mucus, owing to the pH-responsive drug release mechanism of CS than the uncoated-NPs. The amino groups of CS shrink at basic pH due to its deprotonation causing a slow release of FVR at pH 7.4. In contrast, an acidic pH below the isoelectric point (pH 6.5) of CS triggers the protonation of the NHe of FVR .Finding the correct balance between the critical factors in optimization, especially the concentration of the CS-coating, is important to maintain its ideal physical characteristics without compromising the mucoadhesive property of the NPs by electrostatic interaction with the mucus. The investigation of the mucoadhesive property of the NPs confirmed that the optimal amount of CS-coating on the FVR-ALG-NPs is still sufficient to elicit muco-interaction which is evident in the significant change in the size and surface charge of the NPs. The oligosaccharide chain of the mucin presents a negatively charged sialic acid terminal residue that enrobes its interaction with the CS layer, hence the change in the ζ-potential to be more negative. This demonstrated that even though the FVR-MCS-ALG-NPs have a negative surface charge after CS-coating, the optimal concentration of CS can still obtain mucoadhesion that is driven by electrostatic interaction with the positively charged amine groups of D-glucosamine molecule of CS with the sialic acid residue of mucin .Moreover, we also demonstrated the sufficiency of the CS-coating to exhibit enhanced transmucosal permeation through PNM. Our result showed that the optimal concentration of CS on FVR-ALG-NPs still demonstrates a 3- and 6-fold faster flux and superior deposition in the PNM compared to uncoated-NPs and free FVR. In other studies, favorable transmucosal permeation is associated with CS through its ability to disrupt tight epithelial junctions . However50 of FVR [Additionally, we have proven the enhancement of the antiviral effect of the FVR-MCS-ALG-NPs by more than 35-fold compared to free FVR towards the PEDV model that acts as a surrogate virus for SARS-CoV-2. A published concept of improving the antiviral activity of FVR by nano-emulsion for coronavirus was hypothesized to effectively interfere with viral adsorption, invasion, and replication . Another0 of FVR . CompariThis study confirmed that FVR could be delivered via intranasal administration to inhibit coronavirus replication, and its activities could be enhanced by encapsulation in MCS-ALG-NPs. The results demonstrated successful optimization of the physical characteristics of FVR-MCS-ALG-NPs by employing BBD-RSM. The optimal FVR-MCS-ALG-NPs notably exhibited high permeation and deposition of FVR in the nasal mucosa which further explains the enhancement of the antiviral activity of FVR in the PEDV coronavirus model. Finally, we have developed an efficient mucoadhesive NP carrier as a new delivery strategy for FVR through intranasal administration in targeting and reducing coronavirus replication in the upper airways.


We compared solution and LA-MC-ICP-MS 87Sr/86Sr intra-tooth profiles from the second and third molars of 5 caribou from the Western Arctic herd, Alaska. Profiles from both methods showed similar trends, reflecting the seasonal migratory movements, but LA-MC-ICP-MS profiles showed a less damped 87Sr/86Sr signal than solution profiles. Geographic assignments of the profile endmembers to the known summer and winter ranges were consistent between methods and with the expected timing of enamel formation but showed discrepancy at a finer scale. Variations on LA-MC-ICP-MS profiles, consistent with expected seasonal movements, suggested more than an admixture of the endmember values. However, more work in understanding enamel formation in Rangifer, and other ungulates, and how 87Sr/86Sr daily intake translates into enamel are needed to assess the real resolution that can be achieved with LA-MC-ICP-MS.Strontium isotopes analysis is a powerful tool in the study of past animal movements, notably the sequential analysis of tooth enamel to reconstruct individual movements in a time-series. Compared to traditional solution analysis, high resolution sampling using laser-ablation multi-collector inductively coupled plasma mass spectrometry (LA-MC-ICP-MS) has the potential to reflect fine scale mobility. However, the averaging of the  Studies have analysed faunal remains to infer trade and exchange networks3, and other aspects of past economies and societies, including transhumance, animal husbandry and herding practices5. Other archaeological and palaeontological studies have focused on wild taxa to explore seasonal animal palaeobiogeography, including the reconstruction of past migratory behaviours in extinct species7 and important prey-species, as a means to better understand the movement patterns, landscape use and subsistence decisions of human groups that depended on them11.Strontium isotope analysis has proven to be a powerful tool for the reconstruction of past movements. Based upon the premise of a relationship between the strontium isotope chemistry in underlying local geology/local soils and skeletal tissues, these techniques are increasingly employed to investigate human and faunal movements in the past84Sr, 86Sr and 88Sr, and as a radiogenic isotope, 87Sr, which is the product of the radioactive decay of 87Rb (Rubidium). Because of the decay of 87Rb, ancient Rb-rich rocks such as potassium feldspar containing granite and rhyloite, are expected to have high 87Sr/86Sr while more recent lithologies and/or those poor in Rb like carbonates, have a lower ratio12. Via soil formation, strontium becomes available for plants, along with other inputs  which can influence ‘bioavailable’ 87Sr/86Sr. Despite these other influences, the spatial distribution of the bioavailable 87Sr/86Sr across the landscape is mainly related to the nature and age of underlying lithological units14. Bioavailable 87Sr/86Sr is incorporated to mammalian tissues through food and water consumed. During the transfer process from bedrock to animal tissues, 87Sr/86Sr is subjected to negligible fractionation15 and thus, values observed in animal tissues should directly reflect the local bioavailable 87Sr/86Sr of the area where the animal fed during the tissue formation13, albeit with some potential influences of differential digestion or other aspects of food selection or preparation18.A recent effort has been made to produce local and global 87Sr/86Sr isoscapes based on intensive sampling of soil and biological material and/or modelling20. As 87Sr/86Sr varies in bedrock on a geological time scale, modern strontium isoscapes are relevant to explore past mobility, although anthropogenic influences that also affect environmental 87Sr/86Sr  should be taken into account21.Strontium exists naturally as three stable isotopes 12. While bones are constantly renewed during the life of an individual, tooth enamel does not remodel once mineralized and, due to its structure, is particularly resistant to diagenesis22. Thus, 87Sr/86Sr values in tooth enamel can provide reliable geolocation information for individuals during their early stages of life. Studies have proven their usefulness to assess faunal mobility23, relying on the principle that mammalian tooth enamel forms incrementally from the cusp of the crown to the enamel-root junction, according to a two steps process24. During the secretion phase, a protein-rich matrix is produced following a secretion front toward the cervix of the tooth. Then, during the maturation phase, most of the mineralised material is incorporated to the enamel matrix24. This phase is complex and non-linear with a rapid initial mineralization rate that slows down progressively, spanning over weeks depending on the species25. However, mineralization broadly progresses along the growth axis following the secretion front26, allowing temporal inferences on the isotopic signal observed in the tooth. Comparison of bulk enamel 87Sr/86Sr with local 87Sr/86Sr allows to discriminate between local and non-local individuals27 but the analysis of intra-tooth 87Sr/86Sr values can highlight short-term mobility22.In mammals, after absorption in the gut, strontium is incorporated into bioapatite in bones and enamel through ionic substitution of calcium28. This is followed by analysis via thermal ionisation mass spectrometry (TIMS) or by multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS). Analyses can be done on bulk (whole tooth) enamel samples or on samples taken along the growth axis of an individual tooth29. Strontium isotope analysis can be combined with ẟ18O analysis to identify which sections of the tooth formed during cooler or warmer periods, placing the 87Sr/86Sr time-series data within a seasonal framework29. 87Sr/86Sr values generated can then be compared with strontium isoscapes, directly or using probabilistic approaches, in order to identify potential areas of origin or seasonal ranges31. A major drawback of solution analysis is the amount of material needed to conduct the analysis (∼ 1–5 mg) which averages 87Sr/86Sr intakes over weeks32. Moreover, this approach is destructive, limiting its application on rare archaeological and palaeontological specimens.The traditional approach to analyse strontium isotope ratios in tooth enamel is to cut a sample  then to undertake anion exchange chemistry (solution method) to isolate and purify the strontium prior to analysis. During this process the sample is dissolved in high-purity acid, and Sr is isolated using a Sr Resinin-situ measurement using laser-ablation (LA)-MC-ICP-MS, allows high resolution sampling along the enamel surface with often less obvious alteration of the material32. While LA-MC-ICP-MS has been reported to produce less precise and less accurate 87Sr/86Sr data than solution analysis due to molecular/analytical 87Sr interferences stemming primarily from the formation of 40Ca31P16O33, the associated analytical errors can remain low enough to study geographic origin and mobility in many geological/environmental context34. Due to the high sampling resolution of LA-MC-ICP-MS, questions have arisen about its ability to reflect mobility habits on a more refined scale and the potential time resolution that can be achieved37. A main concern is to what extent the 87Sr/86Sr is averaged within the body before and during the mineralization process, regardless of how high-resolution sampling strategies might be. For example, in addition to the daily strontium intake , strontium is released during the continuous remodelling of bones contributing to blood strontium38, and inducing a 87Sr/86Sr baseline reflecting the average 87Sr/86Sr in bones. In ungulates most of the mineralization at any specific location occurs in only a few days before slowing down and then continuing over several weeks25. The real impact of these two averaging processes on the ability of intra-tooth 87Sr/86Sr profiles to reflect changes in location is unclear. Montgomery et al.35 suggested that intermediate values within any intra-tooth sequence are only the product of mixing the signal of the endmembers, the 87Sr/86Sr value of the new location overprinting gradually the original signal. However recent studies on human39 and sheep36 dental enamel have provided evidence of short-term variations in intra-tooth 87Sr/86Sr profiles that contradict an admixture of endmembers signal, and instead reflect more the movement of the individuals through the strontium landscape36. More studies are needed to fully assess the potential of LA-MC-ICP-MS in reconstructing faunal movements, notably in regard to the well-established solution analysis.Alternatively, Rangifer tarandus) teeth from migratory animals, here we aim to compare solution and LA-MC-ICP-MS 87Sr/86Sr intra-tooth profiles from the same individuals. Caribou and reindeer are important taxa in northern environments, and both are central in the culture and subsistence of circumpolar indigenous communities42. There is a long history of Rangifer population exploitation by human societies, in North America, and throughout Eurasia. During the Late Pleistocene in north-west Europe, reindeer were at the centre of the economy and a herd’s seasonal movements likely shaped human subsistence practices and landscape use , and even the cultures and worldviews of human groups43. Modern wild caribou demonstrate variability in their seasonal movements with resident and migratory ecotypes40 and within a given population, migration patterns can drastically change over a period of only a few years44. The extent to which there were diverse ecotypes within Late Pleistocene Europe, and/or the extent to which these behaviours were plastic or conservative through time remains poorly characterised. Reconstructing past mobility and seasonality of reindeer is therefore essential to understand landscape use and other cultural aspects of past human and hominin groups10, but also to understanding the palaeoecology of these species. The increased resolution offered by LA-MC-ICP-MS may help us better understand these. Furthermore, the usefulness of spatial assignment tools in empirically identifying summer and winter ranges from intra-tooth data (generated via solution or LA-MC-ICP-MS approaches) has yet to be tested on a modern, wild migratory Rangifer. The objectives of this study were therefore: (1) to generate novel intra-tooth 87Sr/86Sr data from five modern caribou from the Western Arctic herd, Alaska, a herd of well-documented seasonal movement pattern47, using LA-MC-ICP-MS, undertaken as a continuous raster down the full growth axis, (2) to compare the obtained LA-MC-ICP-MS intra-tooth 87Sr/86Sr profiles to already published intra-tooth profiles from the same individuals, generated by the ‘traditional’ strip sampling and solution method23, and (3) to compare the potential summer and winter range use through the spatial assignment of data from seasonal endmembers obtained from both the solution and LA-MC-ICP-MS sequences.Using modern caribou (87Sr/86Sr intra-tooth profiles from the second (M2) and third molar (M3) of 5 caribou from the Western Arctic herd  to the youngest part (enamel-root junction—ERJ) of the M2 and the opposite trend was observed on the M3  than for solution  and maximum 87Sr/86Sr values were significantly higher . However, median 87Sr/86Sr values in LA-MC-ICP-MS (0.7103 ± 0.0009 SD) and solution profiles (0.7103 ± 0.0009 SD) were similar . LA-MC-ICP-MS profiles had a higher amplitude than solution profiles  and differences in amplitude between LA-MC-ICP-MS and solution profiles were positively correlated with LA-MC-ICP-MS amplitude .Using LA-MC-ICP-MS analysis, we generated erd Fig. . These pM3 Table . For 4 i M3 Fig. . For oneles Fig. . Solutio87Sr/86Sr intra-tooth profiles, we relied and on the ẟ13C and ẟ18O intra-tooth profiles from the same individuals  were similar between LA-MC-ICP-MS and solution analyses  but summer endmembers  87Sr/86Sr values from LA-MC-ICP-MS analysis were significantly higher . Such as for the total amplitude of the profiles, amplitude between endmembers from LA-MC-ICP-MS profiles were higher than in solution profiles .To set seasonality in ls Figs. –S2, asseues Fig.  and highM3 Figs. –S2, with2 and M3 endmembers to determine potential summer and winter ranges, using the assignR package53 in R54 with the global bioavailable 87Sr/86Sr isoscape from Bataille et al.14 cropped to north-west Alaska  with extreme values of 0.7062 and 0.7271  were assigned to the southern range except for the M3-OS of two individuals, both for solution and LA-MC-ICP-MS profiles  were mostly assigned to the known summer range in the north  and assessed how differences in endmembers 87Sr/86Sr values of the M2 and M3 affected the summer and winter geographic assignment of these individuals. Among the 5 individuals, 4 displayed variability in their 87Sr/86Sr intra-tooth profiles, reflecting a migratory behaviour. Direct comparison of the output of the solution and LA-MC-ICP-MS methods requires samples taken at the same location on the tooth, accounting for the larger sampling site for solution analysis26. Here, the initial solution samples were destroyed during the analytical process23. When possible, we used the same tooth, on the lingual surface but for several teeth we had to rely on the molar from the other mandible. Crown height may differ between the inner and outer surface of a tooth, and between teeth of the left and right mandible. Left and right teeth can also present different levels of attrition56. Consequently, samples taken in different teeth or tooth surface should reflect an equivalent but not identical period of time. Thus, we limited our comparison between solution and LA-MC-ICP-MS profiles to global trends and to endmembers values.We compared the solution and LA-MC-ICP-MS 87Sr/86Sr intra-tooth profiles was on the amplitude between endmember 87Sr/86Sr values, solution profiles showing a dampening of the signal compared to LA-MC-ICP-MS. Due to the long mineralisation phase, the isotope input signal is time-averaged during amelogenesis leading to a natural attenuation of the signal within the tooth57. The strength of the attenuation is proportional to the maturation length, with greater attenuation for a longer time of maturation24. In the case of strontium, baseline blood values influenced by the long-term remodelling of bone also have the potential to mitigate the input signal38. Difference in attenuation between solution and LA-MC-ICP-MS analyses is likely due to sampling strategies58. Solution analysis was done on 1.5 mm wide strip samples taken on the entire thickness of the enamel. Enamel matures at different rates in different parts of the crown, with a slower rate of maturation close to the enamel-dentine junction57. Solution samples are likely to have a more extensively ‘averaged’ 87Sr/86Sr signal over a greater section of enamel than LA-MC-ICP-MS samples, spanning a longer maturation period, leading to a stronger dampening58. Moreover, the amplitude of LA-MC-ICP-MS 87Sr/86Sr variations were more consistent with the variations observed on the bioavailable 87Sr/86Sr isoscapes within the WAH range. Only for WACH-0.120, an individual that did not display migratory behaviour in either intra-tooth, do the solution and LA-MC-ICP-MS profiles demonstrate no discrepancy from one another. This suggests that when individuals are exposed to a constant, stable, bioavailable 87Sr/86Sr during the whole period of tooth growth both approaches should lead to similar output with no evidence of attenuation. Methods have been developed to retrieve the input signal from the observed intra-tooth profile for ẟ13C and ẟ18O accounting for attenuation57, and such approaches could be used for 87Sr/86Sr. However, it requires a good knowledge of the mineralisation process, notably of the apposition and the maturation length. Such parameters are known for several hypsodont ungulates  but far less studied in brachydont ungulates61 and remain poorly characterised for Rangifer.The first difference observed between the solution and the LA-MC-ICP-MS 87Sr/86Sr intra-tooth profiles followed similar trends to solution profiles with a decline in 87Sr/86Sr signal while progressing toward the ERJ for the M2 and an increase for the M3, in the four migratory individuals, and a stable, low signal in the likely sedentary individual. Geographic assignments from LA-MC-ICP-MS and solution analyses appear to confirm the interpretation made on the solution 87Sr/86Sr intra-tooth profiles in the original study23. Relying on geological maps to interpret the variation in solution 87Sr/86Sr intra-tooth profiles, the decline in 87Sr/86Sr values observed in the M2 in this first study was attributed to an autumn seasonal movement from the summer range in the north within a high 87Sr/86Sr geological context, relative to this part of Alaska, toward the winter range in the south, within a lower 87Sr/86Sr geological context23. The opposite was observed on the M3, echoing the spring migration. In the current study, broad scale assignment to the known seasonal ranges using solution and LA-MC-IC-MS endmembers led to the same conclusions. However, on a finer scale, through the use of the assignment maps, differences on the potential location within a given seasonal range became evident between the two approaches. For the M2, the OS endmembers of the 87Sr/86Sr profiles were assigned to the summer range for all migratory caribou, while the ERJ endmembers were assigned to the winter range for all but one individual. For their M3, the OS endmembers were assigned to the winter range for half of the caribou and the ERJ endmembers of 3 individuals were assigned to the summer range and one belonged to the winter range. The sedentary individual, WACH-0.120, was always assigned to the southern range. Assignments were more nuanced when looking at the assignment maps. While low 87Sr/86Sr values (< 0.710) clearly designate the use of the southern range in winter, values between 0.710 and 0.711 could either indicate the southern and the northern areas. Higher 87Sr/86Sr values (> 0.7125) were mostly assigned to the Brooks Range in between the summer and the winter ranges. Migratory caribou are highly philopatric to their calving ground62 but show less fidelity to their winter range63. Caribou from WAH mainly winter south of the Brooks Range but can use areas further north with a higher 87Sr/86Sr signature63. Late summer movements are also highly variable in WAH with caribou using the northern range but also the Brooks Range63 within areas of medium to high bioavailable 87Sr/86Sr. OS endmembers should be taken with caution as they may have been exposed to wear as suggested notably by caribou WACH-2000, which had the lowest crown height. In this case the high 87Sr/86Sr value at the beginning of the M3 might not reflect the winter range 87Sr/86Sr signature, but rather a slightly later period and thus the transition toward the summer range. Despite a different degree of attenuation, solution and LA-MC-ICP-MS approaches appeared to reliably discriminate between potential seasonal ranges. However, to go beyond the assessment of broad scale origin a more refined sampling strategy, not normally possible for solution analysis due to sampling size requirements, is needed.Despite differences in endmember values, LA-MC-ICP-MS 87Sr/86Sr intra-tooth profiles showed peaks and troughs not visible on solution profiles. While transition from one endmember to the other occurred gradually on solution profiles, LA-MC-ICP-MS analysis highlighted clear sigmoid trends with steeps changes in 87Sr/86Sr values. Several teeth recorded small-scale increasing and declining phases within the global trend of the profile. Comparing 87Sr/86Sr cow’s molar profiles from solution-TIMS and LA-MC-ICP-MS analyses, Montgomery et al.35 concluded that intermediate values were the result of endmembers admixture and as such the higher resolution of LA-MC-ICP-MS analyses cannot be used to infer fine-scale movements. This was attributed to the long-term time-averaging mineralization process, meaning that a sampled area of tooth will incorporate strontium from multiple and not necessarily entirely synchronous events, and that during maturation strontium may even be incorporated more randomly in the crown, mitigating the signal35. However, measurements on human teeth have shown that minimum and maximum 87Sr/86Sr values from several LA-MC-ICP-MS profiles were outside of the range of the values observed at the beginning and at the end of these profiles, which does not support a simple admixture model of those endmembers26. This has also been observed in the current study. These differences may be attributed to the different tooth types of the species involved, and the different rates of enamel formation and mineralisation. While maturation of the enamel at a given location of a tooth can span a number of months, much of the process occurs in a short period of time before slowing down25. Thus, the resolution that can be achieved with LA-MC-ICP-MS analysis should likely depend on the duration of this fast mineralisation phase. In Mongolian caprines, relative changes in LA-MC-ICP-MS 87Sr/86Sr intra-tooth values reflected the use of pastures for relatively short periods of 3–4 weeks, indicating that for that species it may be possible to capture monthly movements36. Migratory caribou are a highly mobile species that can migrate over hundreds of kilometres, and are continuously moving, except for the wintering period and, for females and neonates, during calving64. High movement rates, particularly during migration, that could cover isotopically diverse areas over a short time frame may greatly limit the ability of the amelogenesis to capture that environmental variation in bioavailable 87Sr/86Sr compared to a singular ‘on–off’ movement between two isotopically distinct ranges. However, in the case of the WAH, caribou take several weeks to migrate across the Brooks Range63, an area separating the winter and the summer ranges and which presents the highest 87Sr/86Sr values in the WAH annual range , would be advantageous in more effectively linking intra-tooth oxygen and strontium profiles. Other approaches, such movement and habitat suitability models used in modern animal ecology to predict seasonal landscape use and migration routes from environmental features69 could also have potential to improve inferences to be made from intra-tooth isotopic datasets from ungulates. A recent study combined high resolution sampling of 87Sr/86Sr with movement modelling to reconstruct the lifetime movement of a mammoth from its tusk7. Applications could be extended through the determining of potential migration routes between seasonal ranges identified from intra-tooth profiles through the incorporation of landscape features that constrain or ease movements. Through accounting for bioavailable 87Sr/86Sr variations in the landscape during migration, the integration of such approaches with intra-tooth LA-MC-ICP-MS data from archaeological and palaeontological specimens would greatly improve our ability to reconstruct the mobility of past animals and—through this—better understand past human societies.Consistent with recent studies on sheep2 in north-west Alaska. Main wintering areas are located south of the Brooks Range in the Nutalo Hills and Seward Peninsula  from 5 individuals  provided by the Alaska Department of Fish and Game. Animals were part of the telemetric survey of the WAH23 and died from natural causes or harvested for purposes unrelated to this study. Each individual was estimated to be 5–9 years old and died within 10 years of each other but were unlikely to come from the same cohort. Following Animal Care policies, the sampled caribou were not equipped with radio-collars before they were at least 2 years old and after teeth formation. Thus, individual telemetric data cannot be used to infer 87Sr/86Sr intake and to correlate it with 87Sr/86Sr intra-tooth profiles, instead movements are inferred from the wider movements of the herd as a whole. Solution analysis of the M2 and M3 was done in a previous study23 and the LA-MC-ICP-MS analysis was conducted on the same teeth where possible, or from the opposing tooth where this was not possible . LA-MC-ICP-MS analysis was conducted on the lingual surface, as opposed to the buccal surface for solution analysis, for the same reasons. Teeth presented different degrees of wear with crown height varying between 7.1 and 10.9 mm for the M2, and between 8.1 and 10.9 mm for the M3.We analysed teeth  to correspond to the late summer/autumn period, and the endmember close to the enamel-root junction (ERJ) to correspond to the wintering period. For the M3, OS and ERJ endmembers were expected to correspond respectively to the winter and to the late summer/autumn periods.In order to infer the seasonal variations in 23. To summarise, the second and third molars (M2 and M3) were extracted from the mandibles, brush-cleaned with water and left to dry overnight. The whole teeth were mechanically abraded to remove surficial enamel. Sampling was done on the buccal face of the anterior loph as it presented a thicker enamel, with the face removed and cleaned from adhering dentine using a tungsten carbide burr. Enamel faces were then marked for horizontal sectioning at ∼ 1.5 mm intervals, ultrasonicated in deionize water  and dried, before being cut into strips using diamond-coated superfine circular drill bits. Samples were then individually ultrasonicated in DI H2O, dried and split longitudinally with ∼ 5 mg of enamel being reserved for 87Sr/86Sr solution analysis and the remainder being retained for carbon and oxygen isotope analysis. In that study, sections were numerically assigned from the ERJ to the OS .For the solution data, sampling and isotope analysis was done in a prior study and is detailed in Britton et al.28 described in detail in Copeland et al.34. The following description of the analytical procedure is reproduced from Britton et al.23, and is also presented there in full. The ∼ 5 mg samples were dissolved in 1 ml 14.3 M high purity HNO3 then evaporated to dryness. The obtained residue was then re-dissolved in 1 ml 3 M HNO3 before loading into pre-conditioned columns containing Sr Resin , being passed through three times. Strontium was then eluted using ultrapure deionized water (18.2 MΩ), dried and re-dissolved in 3% HNO3 and analysis of 87Sr/86Sr ratios was undertaken using a Thermo Fisher Neptune™ (MC-ICP-MS). All the acids solutions used in the procedure were purified through a PicoTrace double-distilled sub-boiling distillation system. The subsequent 87Sr/86Sr measurements on standards and samples were corrected for interferences from krypton (Kr) and rubidium (Rb) and normalized for instrumental mass bias to 88Sr/86Sr = 8.375209 . Analysis of the international strontium isotope standard NIST SRM987  during each analytical session was used for external normalisation of data  (n = 97)). All 87Sr/86Sr values reported here were adjusted so SRM987 = 0.71024072, involving a data correction factor of − 0.00002. Strontium concentrations of the enamel samples were determined using the method described in34, which is accurate to within ± 31 ppm.Strontium was isolated from enamel in clean laboratory facilities at the Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology (MPI-EVA), Leipzig, Germany using a modified version of the method from Deniel and Pin2O for five minutes and then dried in an air extraction hood at room temperature overnight.Enamel samples intended for analysis via LA-MC-ICP-MS were cut from whole teeth using diamond-coated superfine circular drill bits as sections encompassing the whole height of the crown just below the ERJ to the OS. The use of diamond coated drill bits serves to produce both a flat and partially polished cut surface. Resulting sections were then ultrasonicated in DI HStrontium isotope data were measured from the samples using a Coherent GeoLas 193 nm excimer laser ablation (LA) system coupled to a Thermo-Fisher Neptune™ (MC-ICP-MS) located at the MicroAnalysis Facility in the CREAIT Network, Memorial University of Newfoundland, Canada. Up to four samples at a time were mounted planar (laying flat with enamel and adjacent dentine facing up and exposing the full height of the crown in cross-section) on tack in the laser’s sample chamber Fig. . The ana73) and monitoring for the formation of LREE-oxides as a general proxy for oxide formation. Nitrogen was added to the Ar sample make-up gas to break down/suppress interferences74. The potential polyatomic interferences on the strontium isotopes were managed by this combination of low oxide tuning and nitrogen addition. Doubly charged REEs were assumed to be a negligible interference since 89Y was not present in any significant amount. The signal intensity of 88Sr ranged from 3 to 8 V for the samples. First, the measured values were corrected for Kr using a gas blank subtraction. Second, 87Sr was corrected for Rb assuming the natural abundance of 87Rb/85Rb and a correction for mass bias using the mass bias of Sr. Since Rb/Sr was low in these samples, this model of Rb correction was sufficient. Finally, the 87Sr/86Sr values were normalized for instrumental mass bias to 88Sr/86Sr = 8.375209 . 84Sr/86Sr was also calculated for each sample (average = 0.0562 ± 0.004 SD) and compared to the invariant 84Sr/86Sr = 0.0565 as a quality control check. An in-house standard  was also measured in each session. The average 87Sr/86Sr = 0.70922 ± 0.00006 SD (n = 7) was compared to 87Sr/86Sr = 0.7092 for present-day seawater as a check for accuracy of the laser data.Typical operating parameters of the LA-MC-ICP-MS are given in Table 87Sr/86Sr values a running average using a window of 20 data points to obtain the corresponding LA-MC-ICP-MS profile. We then extracted from the solution and LA-MC-ICP-MS profiles the minimum, maximum and median 87Sr/86Sr values. We also considered endmember 87Sr/86Sr values, i.e., the samples closest to the OS and to the ERJ in each profile35. Due to the low number of samples, we conducted non-parametric Wilcoxon signed-rank test to compare the different metrics assessed from solution and LA-MC-ICP-MS 87Sr/86Sr intra-tooth profiles.For each tooth we calculated from the raw LA-MC-ICP-MS 87Sr/86Sr values of both teeth analysed via the two different methods (solution and LA-MC-ICP-MS) using the assignR package53 in R software (v4.2.1)54. The assignR package uses Bayesian spatial assignment to infer the geographic origin of samples from 87Sr/86Sr isoscape. We used the global bioavailable 87Sr/86Sr isoscape published by Bataille et al.14 and the associated spatial error raster. This isoscape is based on extensive collection of field sampling of plants, soils and water, the data being drawn from a variety of studies published over the last decade . Endmembers were treated as samples of unknown origin, and we generated a posterior probability surface, rescaled to 1, for each sample. For both solution and LA-MC-ICP-MS values of a given endmember, we extracted the 20% of the posterior probability surface with the highest probability of origin and calculated the overlap between the two assignment surfaces. Finally, we used the oddRatio tool from assignR to compare with odd ratio the posterior probabilities between summer and winter ranges and assessed from which seasonal ranges endmembers were more likely to originate from (odd ratio = probability of assignment summer range/probability of assignment winter range). Shapefiles of summer and winter ranges were obtained from the Alaska Center for Conservation Science55 and are based on the telemetric survey of the herd by the Alaska Department of Fish and Game between 2004 and 2014.For the 5 individuals, we performed geographic assignment of the endmember Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


A laeA deletion (ΔlaeA) Ganoderma strain exhibited significantly reduced concentration of ganoderic acids. qRT-PCR analysis further revealed that the transcription levels of genes involved in the biosynthesis of ganoderic acids were drastically lower in the ΔlaeA strain. Moreover, deletion of laeA resulted in decreased accumulation of intermediates and abundances of asexual spores in liquid static culture of G. lingzhi. In contrast, constitutive overexpression of laeA resulted in increased concentration of ganoderic acids. These results demonstrate an essential role of LaeA in the regulation of ganoderic acid biosynthesis in Ganoderma.The methyltransferase LaeA is a global regulator involved in the biosynthesis of secondary metabolites by ascomycete fungi. However, little is known of its regulatory role in basidiomycete fungi. In this study, the  Ganoderm lingzhi is a well-known medicinal fungus that has been used to improves health and prevent human diseases for over 2000 years  has been demonstrated to be involved in regulating the biosynthesis of numerous secondary metabolites in ascomycete fungi like achiatum . Howeveroduction . A LaeA anoderma . HoweverGanoderma for the first time. Targeted deletion of laeA significantly reduced GA production. Moreover, the expression of GA biosynthetic genes, accumulation of intermediates, and the abundance of asexual spores also decreased in the ΔlaeA Ganoderma. Further analysis revealed that constitutive overexpression of laeA increased the production of GAs in Ganoderma.Here, we show that LaeA positively regulates GA biosynthesis in the basidiomycete G. lingzhi pJW-EXP-intron-opCas9 . The two sgRNA cassettes were transcribed in vitro using a HiScribe™ T7 High Yield RNA Kit  and purified using the RNA Clean and Concentration™ − 25 Kit .Two sgRNA cassettes , includilaeA sequence was amplified from G. lingzhi genomic DNA using the primers gpd-LaeA-F: 5′-ttcatccccctctcaacATGGCCATCGAATACGCCCG-3′ and ter-LaeA-R: 5′-ctctctgacccgctcat CTACAGGCGGCGCGCGT-3′. The amplified PCR product was fused with the plasmid pJW-EXP  to produce the PJW-EXP-LaeA plasmid.The  pJW-EXP  that wasG. lingzhi protoplasts was conducted as previously described (gpd) promoter and laeA using the primers gpd-F: 5′-CGAGTGACGCAGGTGGTGAC-3′ and ter-R: 5′-GCAGTCGCACAATCTAGCCCT-3′. To screen the ΔlaeA mutants, transformants were picked from CYM selective plates containing 250 mg/l hygromycin B after genetic transformation of G. lingzhi (the pJW-EXP-intron-opCas9 strain) protoplasts with the pJW-EXP-ophph plasmid , lanosterol synthase gene (ls), laeA, and gl25098 were then determined with the cDNA pools by qRT-PCR, as previously described  and reverse-transcribed using the PrimeScript™ RT reagent kit . The transcription levels of the squalene synthase gene . The protein encoding gene gl27879 that is hereafter referred to as LaeA was identified with 44% to RDB17543 and with a corresponding E value of <1.00E−80. G. lingzhi laeA is 1,379-bp long and has an open reading frame of 1,125-bp that encodes a protein of 375 amino acids. The amino acid residues in the 115–295 region of G. lingzhi LaeA encode an S-adenosylmethionine-dependent methyltransferase domain  was observed in the control strain and transformants 1 and 2, while amplification of an approximately 250 bp band was observed for the transformant 3  and Δn day 12 . The Δla gl25098  were alsectively .G. lingzhi (wild-type strain) protoplasts. Transformants were selected on CYM plates containing 2 mg/l carboxin after three rounds of growth on nonselective CYM plates  in the positive control and transformants 1, 2, 3, and 5 (laeA in the mycelia of the wild type (WT) and the transformant 1 strains. laeA was overexpressed in transformant 1 under liquid static culture conditions. Further, the transcription levels of laeA in transformant 1 were 5.0-, 4.1-, and 5.5-fold higher than those of the WT strain on days 3, 6, and 9, respectively  and the Yunnan Applied Basic Research Project (No. 2018FB065). J-WX also thanks to the Yunnan 10,000 Talents Plan-Young and Elite Talents Project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Forty-five male Wistar rats, aged 20 months, were randomly divided into five groups: control (C), Swimming High-Intensity Interval Training (HIIT) (S-HIIT), Swimming HIIT with resveratrol supplementation (S-HIIT-R), resveratrol supplementation (R), and solvent of resveratrol supplementation (SR). S-HIIT and resveratrol groups performed the exercise and received resveratrol  for six weeks. Western blot analysis was performed to determine the protein level in the hippocampus. The amount of SIRT5 and SOD1 proteins in the hippocampus increased. S-HIIT with resveratrol or resveratrol alone increased the PDH-E1α level significantly. The amount of NAD+ was analyzed by assay kit that was reduced in S-HIIT, S-HIIT-R, and SR groups compared to controls. The results showed that resveratrol and S-HIIT attenuated the age-related brain changes by increasing the expression of SOD1 and SIRT5 and reducing the level of NAD+ in the hippocampus. Considering these findings, S-HIIT and resveratrol supplementation could be proposed as strategies to attenuate age-related brain changes. Resveratrol alone and exercise through the regulation of crucial proteins and cofactors can influence mitochondrial metabolism and oxidative stress in the hippocampus of aged rats.Mitochondrial dysfunction and increased oxidative stress cause damage to cells which can lead to the aging process and age-related diseases. Antioxidants such as resveratrol and high-intensity exercise can benefit oxidative damage prevention. This study is aimed at evaluating the effects of swimming high-intensity interval training and resveratrol on mitochondrial metabolism key proteins, SIRT5, SOD1, and PDH-E1 Mitotylation .In addition to a decline in the ability of cells to scavenge ROS, aging is linked to increases in the mitochondria's ability to release superoxide anions . It appeα subunit of the PDH enzyme, decreasing pyruvate use in the TCA cycle [α  PDH enzyme and PDH inhibition which consequently restrains acetyl-CoA production of pyruvate in mitochondria [Another essential protein is pyruvate dehydrogenase (PDH), which links glycolysis to tricarboxylic acid (TCA). PDH activity is regulated through reversible phosphorylation in the E1CA cycle . It has chondria , 23.Previous studies have shown that HIIT can reduce hippocampal oxidative stress by decreasing lipoperoxidation and inflammatory markers and increasing BDNF gene expression . It has Moderate-intensity exercise has also been reported to reduce oxidative stress and inflammation , 28. Resα and also the level of NAD+ in the aged rat hippocampus.The hippocampus, a limbic system structure, is affected by aging and mitochondrial dysfunction. It has been also demonstrated that this brain region responds the most to exercise, especially high-intensity interval training (HIIT) . That isn = 9 in each group) and named as control or C group, Swimming High-Intensity Interval Training or S-HIIT group, 1% carboxymethyl cellulose+resveratrol supplementation or SR, S-HIIT+Resveratrol supplementation or S-HIIT-R, and Resveratrol supplementation or R. Twenty-months rats underwent the novel object recognition test to cognitive evaluation (as an aging indication) of rats and also for noninterference of that evaluation with exercise protocol. In addition, the open field test was used to evaluate the lack of motion impairment in 20-months rats. Finally, 45 male 20-months rats (350-450 g) whose novel object recognition test results were negative and had no motion impairment were included in the current study [In this developmental study, 45 male 20-month-old Wistar rats (weighing 400-450 g) were purchased from the Neuroscience Research Center of Kerman University of Medical Sciences. Rats were maintained with controlled periods of the 12:12 h light-dark cycle (lights on 07:00–19: 00 h) at the temperature 22-24°C. All rats had free access to both water and food. Tap water and food pellets were provided ad libitum. Rats were randomly divided into five groups , produced in rabbit), 1/5000 dilution, were then applied (under incubation) to blots overnight in 4oC, and then washed (using TBST) three times and 10 minutes each time, and secondary antibodies (horseradish peroxidase-conjugated anti-rabbit antibody), 1/100,000 dilution, applied for 1-2 hours at 24°C. Membranes were again washed with TBST and incubated to ECL (GERPN2232). The light emitted from the luminescence reaction was recorded on BioMax film (Roche-000000011666657001) in the darkroom. After that, using photo-processing solutions, the images appeared. Film densitometry was determined using Image J software.As previously described, the hippocampus was collected and prepared for western blotting . Samples+ level in hippocampus tissue was determined using the colorimetric method by NAD+/NADH assay kit described by the manufacturer  [+/NADH cycling assay mix. After two hours, the color was developed with NADH developer solution, and the absorbance was measured at 450 nm using a microplate reader. An aliquot of homogenized material was utilized to assess the concentration before ultrafiltration. Before ultrafiltration, an aliquot of homogenized material was used to test the protein concentration using the conventional bio-rad procedure of measuring the absorbency at 595 nm. Based on conventional NADH measurements, the amount of NAD+ was represented in pmol per mg protein.NAD348, UK) . HippocaP value less than 0.05 was defined as significant.All statistical analyses were performed using SPSS . The normality of the distribution of research variables was examined using Shapiro–Wilk test. One-way and two-way analyses of variance (ANOVAs) were used to analyze the data. When a statistically significance difference was found, Tukey's post hoc multiple comparison test was used to determine points of significant difference. All values in the present study were expressed as mean ± SEM, and a The body weight of the rats in all groups in the 1st, 4th, and 6th weeks was shown in P < 0.05). In addition, the SOD1 protein level increased significantly in S-HIIT-R and R groups and significantly decreased in the SR group (P < 0.05) (P = 0.0001) and SOD1 protein (P = 0.0001) between all groups. Tukey's post hoc test results have also shown a statistically significant difference for SIRT5 between SR and S-HIIT groups (P = 0.0001), between R and S-HIIT groups (P = 0.0001), and S-HIIT and S-HIIT-R groups. In addition, there is a statistically significant difference for SOD1 between all intervention groups (P < 0.05) (P = 0.0001). One-way analysis of variance of the present study indicated that average amounts of E1(α) catalytic subunit of pyruvate dehydrogenase enzyme and Tukey's post hoc test in R and S-HIIT-R groups increased significantly compared to SR group   . In resp < 0.05) . The resctively) .α in the SR group, compared to the control group did not have a significant difference (P = 0.99). Moreover, it was also realized that the amount of this subunit in the swimming HIIT group compared to the control group increased but not significantly (P = 0.383). One-way analysis of variance of the current study revealed a statistically significant difference in NAD+ level (P = 0.005) between all groups . Tukey's post hoc test showed a statistically significant difference for NAD+ between S-HIIT and S-HIIT-R groups (P = 0.003).It was also found that the amount of PDH-E1l groups . It has P = 0.0001) (P = 0.0001).Additionally, our results have shown that the amount of average blood lactate level (mMol/L) in the 6th week in the aged rats of the S-HIIT group declined in comparison with the 1st and 3rd weeks ( 0.0001) . In contα in the aged rat hippocampus. The present study showed that resveratrol alone or with swimming high-intensity interval training could increase the SIRT5, SOD1, and PDH-E1α proteins of aged rats' hippocampus. In addition, we found that NAD+ level was reduced in all groups. Also, our results showed that resveratrol with swimming high-intensity interval training increases blood lactate within six weeks. There are differences between the control and solvent of resveratrol groups in the protein levels of SIRT5 and SOD1, which could be due to the gavage-induced stress.This study examined the effects of swimming high-intensity interval training and resveratrol on the protein levels of SIRT5, SOD1, and PDH-E1α in the S-HIIT-R and resveratrol supplementation groups increased significantly. It was also indicated that in the S-HIIT group, the amount of PDH-E1α increased, but it was not significant. Considering these findings, swimming high-intensity interval exercise training and resveratrol may have been proposed as strategies to attenuate age-related brain changes. Evidence is now rapidly emerging showing brain aging of the mammalian is characterized by increased biomarkers of oxidative stress and accumulation of damage to DNA or protein, ultimately leading to neuronal dysfunction [This study demonstrated, for the first time, that swimming high-intensity interval training and resveratrol increases the protein levels of SOD1 and SIRT5 in the hippocampus region. Increasing SOD1 leads to decreasing oxidative damage. In addition, increasing SIRT5 through deacetylation increases the enzymatic activity of carbamoyl phosphate synthetase 1 (CPS1). The amount of PDH-E1function . It has function .Furthermore, it was reported that resveratrol supplementation, by decreasing mitochondrial dysfunction and oxidative damage, maintains its neuroprotective role . These s+ level, was distributed in the mitochondria, cytoplasm, and nucleus in cerebellar granule cells [+ by increasing the SIRT5 protein level contributes to a broad range of age-related pathophysiologies [Resveratrol affects a variety of proteins and signaling pathways . The curle cells . In an ile cells . Besidesle cells , suggestiologies .Consistent with our results, it has been revealed that transgenic expression of SOD1 causes attenuation of the accumulation of the oxidative damage of DNA and protein in the neurons in aged mice  and prevα protein in aged rats' hippocampus, and also due to this fact that aging leads to phosphorylation of E1α subunit of PDH enzyme and consequently to inhibition of PDH complex [α and finally increases the activity of PDH enzyme in mitochondria of rat's hippocampus cells. It was also found that high- and low-intensity combined exercise for eight weeks on an ergometer cycle among 60- to 72-year-old men has increased the amount of PDH-E1α protein in muscular cells [2 reaches a maximum of 60%-70%, and in trained people, the VO2 reaches 75%-80%, the lactate threshold is too much. Therefore, the supralactate threshold, special for high-intensity training, has to be noticed in older people. To this end, many studies have shown that aerobic exercise of 60% to 80% VO2 max is possible and practical [Regarding the results of this study in which the resveratrol supplementation increased the amount of PDH-E1 complex . This, aar cells . What isractical . Also, lractical .Inconsistent with these results, recently, it was demonstrated that HIIT decreased the neural survival and memory functions in the rat hippocampus through the downregulation of dynein and KIF5B, respectively . One rea+ in the hippocampus. Furthermore, resveratrol and S-HIIT as bioenergetic interventions can affect mitochondrial metabolism through PDH-E1α in the hippocampus of old rats. Also, lactate, a by-product of muscle exercise, enters the brain and is used as an energy source. Based on our findings and previous studies, it can be proposed that resveratrol and S-HIIT might improve hippocampal function by influencing these factors.In general, the current study describes some novel effects of resveratrol and S-HIIT on attenuating age-related brain changes by increasing the expression of SOD1 and SIRT5 and decreasing the level of NADIn the current study, the limitations must be acknowledged. First, uncontrollable stress  may be confounding factors. It may create a bias in the data obtained. Second, age-related changes also can be one obvious limitation of such studies. A third limitation is the lack of behavioral evaluations due to the possible confounding role of stress resulting from testing. Also, the animals were not sacrificed until 48 hours had passed since the HIIT. This process would affect the production of factors we measured. Finally, this study was conducted on animals; however, HIIT should be undertaken on the aged population who do not have orthopedic problems in human studies.


Furthermore, protein–protein interaction analysis identified four protein complexes where clusterin and ApoE participated as partner proteins. Thus, J20 U-exo and N-exo contain proteins related to lipid- and Aβ-metabolism in the early stages of AD, providing a new insight into the underlying pathological mechanism of early AD.Alzheimer’s disease (AD) is a common dementia disease in the elderly. To get a better understanding of the pathophysiology, we performed a proteomic analysis of the urine exosomes (U-exo) in AD model mice (J20). The polymer precipitation method was used to isolate U-exo from the urine of 3-month-old J20 and wild-type (WT) mice. Neuron-derived exosome (N-exo) was isolated from U-exo by immunoprecipitation. iTRAQ-based MALDI TOF MS/MS was used for proteomic analysis. The results showed that compared to WT, the levels of 61 and 92 proteins were increased in the J20 U-exo and N-exo, respectively. Gene ontology enrichment analysis demonstrated that the sphingolipid catabolic process, ceramide catabolic process, membrane lipid catabolic process, Aβ clearance, and Aβ metabolic process were highly enriched in U-exo and N-exo. Among these, Asah1 was shown to be the key protein in lipid metabolism, and clusterin, A Alzheimer’s disease (AD) is a common dementia disease in the elderly population. The presence of extracellular amyloid plaques and intraneuronal neurofibrillary tangles in certain brain areas are considered the main pathological hallmarks of the disease . The amypoE) has a crucial role, is suggested to be disturbed . The closer it was to zero, the more significant the enrichment was. Pathways with p < 0.05 were defined as pathways that were significantly enriched. The larger the enrichment factor, the greater the degree of enrichment.We submitted the total proteins identified in U-exo and N-exo to Metascape  within a cluster was selected to represent the cluster to avoid wasting time on redundant enriched terms.To further capture the relationships between the enriched terms, hierarchical clustering analysis on the GO enriched terms was performed. A subset of enriched terms was selected and rendered as a network plot. The enriched terms were collected and grouped into clusters based on their membership similarities. The kappa scores were used as the similarity metric, and sub-trees with a similarity of >0.3 were considered a cluster . The mosp value < 0.001, a minimum count of 3, and an enrichment factor > 1.5 to filter out pathways that were not enriched enough.All pathway enrichment analyses adopted uniform criteria, a Metascape was also used to carry out PPI enrichment analysis with the following databases: STRING, BioGrid, OmniPath, and InWeb_IM. Only physical interactions in STRING  and BioGrid were used. The resultant network contained the subset of proteins that form physical interactions with at least one other member in the list. If the network contained between 3 and 500 proteins, the MCODE algorithm was applied to identify densely connected network components . The MCOhttps://psort.hgc.jp/, accessed on 27 August 2022). The brain expression of the identified proteins was performed by GenAtlas (http://genatlas.medecine.univ-paris5.fr/ accessed on 20 August 2022). The online database CellMarker 2.0 (http://117.50.127.228/CellMarker/ accessed on 10 September 2022) was used to search for neuronal markers. Bar graphs of GO enrichment analysis were generated by an online tool (http://www.bioinformatics.com.cn/ accessed on 11 July 2022) with R package, and dot bubble plots of enriched BP terms were also created by the R package.In addition, the subcellular localization of identified proteins was carried out by the online tool WoLF PSORT (


Kidney transplant recipients showed a weak humoral response to the mRNA COVID-19 vaccine despite receiving three cumulative doses of the vaccine. New approaches are still needed to raise protective immunity conferred by the vaccine administration within this group of high-risk patients. To analyze the humoral response and identify any predictive factors within these patients, we designed a prospective monocentric longitudinal study of Kidney transplant recipients (KTR) who received three doses of mRNA-1273 COVID-19 vaccine. Specific antibody levels were measured by chemiluminescence. Parameters related to clinical status such as kidney function, immunosuppressive therapy, inflammatory status and thymic function were analyzed as potential predictors of the humoral response. Seventy-four KTR and sixteen healthy controls were included. One month after the administration of the third dose of the COVID-19 vaccine, 64.8% of KTR showed a positive humoral response. As predictive factors of seroconversion and specific antibody titer, we found that immunosuppressive therapy, worse kidney function, higher inflammatory status and age were related to a lower response in KTR while immune cell counts, thymosin-a1 plasma concentration and thymic output were related to a higher humoral response. Furthermore, baseline thymosin-a1 concentration was independently associated with the seroconversion after three vaccine doses. In addition to the immunosuppression therapy, condition of kidney function and age before vaccination, specific immune factors could also be relevant in light of optimization of the COVID-19 vaccination protocol in KTR. Therefore, thymosin-a1, an immunomodulatory hormone, deserves further research as a potential adjuvant for the next vaccine boosters. Unfortunately, large series of patients including kidney transplant recipients (KTR) who have already received two doses of mRNA vaccines showed poor seroconversion rates in comparison to the general population related to their immunosuppressive treatment. Different studies have shown the seroprotection rates are under 20% after the first dose, and less than 50% after the second one, while in the general population, these values are close to 100% . AlthougThe distribution of immune cell populations impacts the outcome of immune responses to different viral infections and vaccination settings such as the CD4/CD8 T-cell ratio in HIV-infected subjects , or the 22.1We designed a prospective monocentric longitudinal study of kidney transplant recipients (KTR) receiving three doses of mRNA-1273 COVID-19 vaccine (100 μg per dose) and healthy controls receiving two doses of BNT162b2 vaccine (30 μg per dose). KTR from the Virgen del Rocio University Hospital , were recruited if they received kidney transplant more than one month before the beginning of the study, and were older than 18 years old and signed informed consent to participate. In our region, KTR received the first dose of vaccine in April 2021. Blood samples were collected up to one month before the first dose (T0), 3-4 weeks after the first dose (T1) (i.e. just before the administration of the second dose), one month after the second dose (T2), four months after the second dose (T3) and one month after receiving the third dose (T4)  and run on a DiaSorin LIAISON XL platform . According to the manufacturer’s data, the sensitivity and specificity of this test were 98.7% and 99.5%, showing a good correlation with microneutralization test . Antibody concentration, expressed as BAU/mL, was automatically calculated by the analyzer from AU/mL by the following conversion formula: AU/mLx2.6=BAU/mL. A positive result was considered as ≥33.8 BAU/mL. The levels of IgG antibodies against the trimeric SARS-CoV-2 Spike protein were considered as a continuous variable, corresponding to the magnitude of the humoral response , but they were also transformed into a dichotomous variable, corresponding to the ability to response or seroconversion, defined as an antibody titter higher or equal to 33.8 BAU/mL (the established threshold for the used assay).IgG antibodies against the trimeric SARS-CoV-2 Spike protein were quantified in serum samples by chemiluminescence assay  by standard procedures at the Immunology Service of our Hospital.2.4®).Plasma and serum samples were collected and stored at -80°C until used. Biochemical and inflammation-related biomarkers were determined by standard procedures at Biochemistry Service of the Virgen del Rocío University Hospital, at T0, in serum or plasma samples when proceeded. Quantification of the homocysteine levels were determined by photometry, whereas high sensitivity C-reactive protein (hsCRP) and β2-microglobulin levels were determined by an immunoturbidimetric assay in Cobas 701 system . eGFR was calculated by CKD-EPI index. Tα1 was determined in baseline plasma samples using Human Thymosin Alpha 1 (Tα1) Elisa kit  were isolated from fresh blood using density gradient centrifugation. Then, PBMC were cryopreserved until DNA was extracted by using Omega BIO-TEK, E.Z.N.A blood DNA Mini Kit. The ratio between number of copies of the telomere sequence and the single copy gen Beta-globin was determined. Copy number quantifications were performed by qPCR following a standard protocol . For eac2.6Thymic output was calculated as the sj/β-TRECs ratio by Droplet digital PCR (ddPCR), in a single reaction, optimized from , and acc2.7Statistical comparisons between groups were performed using non-parametric Mann-Whitney U-test for continuous variables or the chi-squared test for the categorical variables. For multiple longitudinal comparisons, the Friedman test applied. Correlations were explored by the non-parametric Spearman´s ρ coefficient. A p-value <0.05 was considered statistically significant. Multivariable linear or logistic regression were performed to determine independent factors affecting to antibody titers or seroconversion rates after vaccination, respectively. Those variables statistically significant in the previous bivariate analysis and those clinically or biologically relevant were included. Statistical analysis were performed using IBM SPSS v21.0 and graphics were generated with GraphPad Prism 8.0 .33.1Eighty-seven kidney transplant recipients (KTR) were initially recruited before receiving the first dose of the COVID-19 vaccine, but only 74 KTR were included for analysis and 54 of them remained at the last time-point (see 3.2p<0.001) and T2   (Seroconversion rates (defined as ≥33.8 SARS-CoV-2 Trimeric-S IgG BAU/mL) were lower in KTR than HC group at T1  Table 1.vs. 44%, p=0.002) and higher antibody levels than before the booster dose . Fifty-four patients remained at the follow-up after the administration of the third dose of mRNA vaccine for the analysis of the humoral response to the booster dose in those patients (3.3p=0.04), as well as higher baseline thymic output, measured as the sj/β-TRECs ratio  (p=0.017). However, the baseline for the telomere relative length in circulating mononuclear cells did not associate with the seroconversion rate . We could measure the pre-vaccination mycophenolate blood level in 38 patients and we found that, non-responders presented higher baseline blood levels of this immunosuppressive drug (4.6 [3.1-6.3] vs. 2.6 [0.8-3.1] p<0.001), and such levels correlated to a lower antibody production   in patients who were treated with mTOR inhibitors in triple combined therapy instead of the mycophenolate-mofetil. No differences were found between these triple combinations and the dual therapy of corticosteroids plus tacrolimus (We observed that 19/19 (100%) of KTR non-responders to the third dose were under mycophenolate mofetil as immunosuppressive therapy compared to 25/35 (71%) among responders (3.5p=0.068) and presented higher baseline levels of β2-microglobulin (p<0.001) (p=0.026) and lower counts of several immune cell populations, such as lymphocytes (p=0.012), B-cells (p=0.010), T-CD3 cells (p=0.025) and T CD4-cells (p=0.010) but higher frequency of neutrophils (p=0.001)  (p=0.210). Regarding correlations, we found negative correlations between the specific antibody titer at T4 and age , proteinuria , ferritin , β2-microglobulin , neutrophils%  and mycophenolate-mofetil blood level , whereas positive correlations were observed between the specific antibody titers at T4 and T3 , baseline eGFR , B-cells/µL  and sj/β TRECs ratio .We obtained a data set comprised of the results from routine biochemical tests and hemograms which can provide insight into the immune response of patients to the vaccination. In addition to the demographical and immunosuppressive therapy related variables, we also analyzed inflammatory markers, kidney function markers and immune cell subsets. These variables are shown in 3.6p=0.011), β2-microglobulin  and T3 antibody titer  were independently associated with T4 antibody titers, whereas CD4 T-cells tended to  (p=0.037) whereas the blood concentration of mycophenolate-mofetil tended to .We performed a multivariable analysis including, besides sex and age, variables related to each of the following categories: demographical parameters, inflammatory-related markers, thymic function-related factors, immune cell subsets, kidney function parameters and immunosuppression therapy. Taking into account that, due to the sample size of our study, we were limited to include up to eight variables in a multivariable analysis, we selected at least one representative variable from each mentioned category, according also to its biological relevance in the vaccination response context. The best fitted model showed that baseline creatinine serum level  Table 3.4In our cohort of kidney transplant recipients (KTR), the humoral response against the COVID-19 vaccine was seriously impaired, even though a third dose/booster significantly improved the seroconversion rate and antibody titers. Not surprisingly, immunosuppressive therapy played a critical role in both, the capability of responding and the humoral magnitude achieved. Remarkably, the capability of responding was associated with baseline thymic-related factors, such as the concentration of thymosin-α1, while the magnitude of the response was associated with different factors, including age and baseline renal and inflammatory status.The KTR cohort showed an inferior response to the COVID-19 vaccine in comparison to the HC group, after two doses and even after an additional third dose, in the case of KTR. Although both groups were not age and sex matched, and were vaccinated with different vaccine mRNA platforms/doses, this result is consistent with previous data on similar cohorts , 19, 20.Due to the risk of developing severe disease in this population  and sincIn this study, we have analyzed immune-related factors potentially involved in the humoral response to vaccination. Interestingly, we found a strong positive correlation between the antibody titers after the two-doses schedule and the booster. Not surprisingly, we observed a poor response in those patients receiving mycophenolate-mofetil as immunosuppressive therapy. Moreover, age was related to lower antibody levels after three doses of vaccination. Both results are in accordance with those from a different KTR cohort receiving two doses of mRNA-1723 vaccine . RegardiA better renal function and lower plasma levels of inflammation-related markers, such as neutrophils and B2-microglobulin, were associated with a better response in our KTR patients. B2-microglobulin is commonly elevated in KTR due to the immunosuppressive status and deteriorated kidney function . FurtherIn this line, we also explored the thymic function, which plays a main role in the maturation and functionality of T-cells and has been outlined in several contexts of limited immune responses, such as HIV-infection, as well as in aging , 31. As We cannot conclude about the clinical protection achieved by the COVID-19 vaccine in our KTR cohort due to the low number of patients and the low rate of infection in our study period. However, from the five documented cases of SARS-CoV-2 infection after the administration of two doses of mRNA COVID-19 vaccine, three of them required hospital admission and one of those patients died due to a bilateral pneumonia. The other two patients presented mild symptoms. This is consistent with the rate of potentially seroprotected subjects in our cohort (50%), that is those patients reaching 260 BAU/mL, a threshold recently proposed for seroprotection , which iWe could not address the cellular response after vaccination in these patients, an important aspect to understand the full dynamics of the immune response after the immunization, and the role of thymic function in such response. Interestingly, an absence of correlation between SARS-COV-2 antibody titers and T cell response in kidney transplant recipients has been described .Our study has additional limitations, such as a low sample size and a large number of variables analyzed which limits the statistical power to conclude about the immune predictors as well as about the role of the combinations of immunosuppressive drugs in the humoral response to COVID-19 vaccine in kidney transplant recipients. In addition, the thymic output data were obtained from PBMCs samples, rather than the isolated T-cell compartment, comprising thymic emigrants, which would therefore have increased the sensitivity of quantification. However, our novel findings could help in the design of future immunization strategies aiming the improvement of the COVID-19 vaccine response in these immunosuppressed patients and suggest the possibility of the use of thymosin-α1 as a vaccination adjuvant to improve their response following the next boosters in this risk population.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Comité de Ética de la Investigación de los Hospitales Vírgen Macarena y Vírgen del Rocío de Sevilla. The patients/participants provided their written informed consent to participate in this study.Patient recruitment and clinical data collection . Quantification of antibody titers (CL). Experiments of immune subsets . Experiments of biochemical and inflammatory markers (AA-R). Processing of samples and experiments of thymic function-related markers . Data base management and statistical analysis . Manuscript preparation . YP conceived the study, obtained funding and supervised the project. All authors contributed to the article and approved the submitted version.


The current attempt was made to detect the amino acid homocysteine (HMC) using an electrochemical aptasensor. A high-specificity HMC aptamer was used to fabricate an Au nanostructured/carbon paste electrode (Au-NS/CPE). HMC at high blood concentration (hyperhomocysteinemia) can be associated with endothelial cell damage leading to blood vessel inflammation, thereby possibly resulting in atherogenesis leading to ischemic damage. Our proposed protocol was to selectively immobilize the aptamer on the gate electrode with a high affinity to the HMC. The absence of a clear alteration in the current due to common interferants (methionine (Met) and cysteine (Cys)) indicated the high specificity of the sensor. The aptasensor was successful in sensing HMC ranging between 0.1 and 30 μM, with a narrow limit of detection (LOD) as low as 0.03 μM. Homocysteine, 2-amino-4-sulfanylbutanoic acid, or HMC is found in human blood as an essential amino acid that is not introduced via food sources but originates from methionine, where the Met present in food is converted to cysteine (Cys) through homocysteine (HMC). The reaction can reverse from HMC to Met; if the reaction stops at HMC, it cannot proceed to Cys and Met, so that the HMC concentration in biological systems may rise above the normal dose of 5–16 μM, which is called hyperhomocysteinemia. As a result, this phenomenon can be associated with many health risks, such as heart attack, osteoporosis, and pregnancy complications ,4.Various techniques were previously used to quantify HMC, some of which are HPLC, GC-MS, capillary electrophoresis, two-photon fluorescent chemosensors, LC-ESI-MS/MS, and LC/MS/MS ,7,8,9,10There is evidence for the use of electrochemical approaches to determine HMC ,40. The The present attempt was made to integrate the merits of the above-mentioned substances and approaches to construct ultra-sensitive electrochemical sensors based on the modification of gold nanoparticles/carbon paste electrode surfaces with aptamers (AP/Au-NS/CPE). Based on the evidence, a facile fabrication of AP/Au-NS/CPE was successful in electrochemically biosensing the HMC. The suggested system is appreciable and reliable for electrochemically biosensing the HMC.2)6 ACCA GCAC ATTC GATT ATAC CAGC TTAT TCAA TTCA CAGC TATG TCCT ATAC CAGC TTAT TCAATT−3′ [The sequences of utilized oligonucleotides  consisted of DNA, 5′SH- while stirring at ambient conditions. Following the PBS washing process, the CPE was positioned in a three-electrode system. The DPV determinations were performed in 0.1 M PBS  at ambient conditions. The voltammetric analysis was performed by PGSTAT 302N Autolab potentiostat/galvanostat (the Netherlands). The conventional three-cell electrode system used contained a CPE work electrode, an Ag/AgCl reference electrode, and a platinum wire counter electrode. A Metrohm 713 pH meter was employed to measure pH values using a glass-reference electrode. To do DPVs, the CPE was placed in a constant concentration of HMC (5 mL) and 0.1 M PBS  was blended with nujol oil (0.3 mL) in a glassy mortar. After preparing the carbon paste, it was placed inside the electrode hole and smoothed with a filter paper to obtain a shiny appearance. 4 + 0.1 M KNO3) at −400 mV for 400 s (Au-NS/CPE). The Au-NS/CPE surface was coated with a 4.5 μM aptamer solution (5 μL). The electrode was subsequently kept vertically in a moist chamber overnight for self-assembly. As-prepared Au-NS/CPE was positioned in 1 mM 6-mercapto 1-hexanol within 60 min after washing with 0.1 M PBS (pH = 7.0) to limit common sites and to obtain the aptamer strands in straight coordination. The washing process for the electrode in each step was performed via PBS . The CPE surface was modified with Au-NS by immersing the electrode inside the gold solution  was immersed in the HMC solution (2 mL) with variable PBS concentrations  for 60 min. After washing, the electrochemical detection was applied to agglomerated HMC in a 25 mL buffer , exploiting the differential pulse voltammetry (DPV). The FE-SEM images of Au-NS modified CPE at different magnifications are shown in The AP/Au-NS/CPE sensor was examined for its behaviors in terms of buffer concentration, aptamer concentration, type of buffer, and time of interaction, followed by their optimization in a solution containing HMC (0.1 μM).In The impact of PBS concentration (ranging from 0.02 to 0.2 M) on HMC detection was explored in solutions carrying 0.1 µM HMC spiked in PBS, the results of which are shown in Under the optimized circumstances, the best accumulation time, the best buffer, and the best buffer concentration were selected to be 65 min, phosphate buffer, and 0.1 M phosphate buffer for future research. The standard curve of HMC determination was plotted using as-obtained AP/Au-NS/CPE under optimal circumstances. The solution HMC was detected by its oxidation peak on the modified electrode. Thus, the DPV was recorded for variable HMC concentrations . The peaThe practical applicability of an aptasensor was explored by sensing HMC in real specimens of human urine and serum samples in accordance with the standard addition method. The preparation of biological specimens was carried out according to previous reports . The resA comparison was made for the HMC aptasensor components, by using available electrochemical approaches in terms of accuracy and validation. Clearly, the LOD ranges are better or more linearly proportional to those listed in The current attempt was made to detect the amino acid homocysteine (HMC) using an electrochemical sensor functionalized with aptamers (aptasensor). The proposed aptasensor was examined for its analytical performance under optimized circumstances. It was easy to use and cost-effective. We explored the influences of the concentration of buffer, concentration of aptamer, type of buffer, and time of interaction. The linear dynamic range at a working potential of 520 mV (versus Ag/AgCl) was from 0.1 to 30.0 µM under the optimized circumstances, with the limit of detection as narrow as 0.03 μM. Further, the practical applicability of the sensor was confirmed by sensing HMC in real biological specimens.